Prosthetic kidney and its use for treating kidney disease

ABSTRACT

The invention is directed to a prosthetic kidney, to methods of making a prosthetic kidney and to methods of treating kidney disease with a prosthetic kidney. The prosthetic kidney comprises nephron analogs on the exterior surface and an enclosed porous membrane structure equipped with an effluent channel for collecting and draining urine from the device. The nephron analogs are prepared by implanting a device containing renal tubule analogs on the membrane structures and inducing angiogenesis to form glomeruli-like structures. The renal tubule analogs are prepared by seeding kidney cells on the porous membrane structure and culturing this composite in vitro.

This application claims the benefit of provisional application60/025,511 filed Sep. 5, 1996.

BACKGROUND

1. Field of the Invention

The invention is directed to a prosthetic kidney, to methods of makingthe prosthetic kidney and to methods of treating kidney disease with theprosthetic kidney.

2. Description of the Background

The kidneys remove metabolic wastes from the blood, control fluidbalance by maintaining homeostasis, and provide important regulatoryactivities by secreting hormones. Normally about 20% of the blood pumpedby the heart is treated by the kidneys.

Nephrons, the functional unit of the kidneys, treat blood by threeprocesses: filtration, reabsorption, and secretion. Each kidney containsabout one million nephrons, each consisting of a renal corpuscle and arenal tubule. The shape of a nephron resembles a miniature funnel with avery long convoluted stem. Blood enters the renal corpuscle, through theglomerulus. The filtrate from the blood enters the glomerular capsule,also called Bowman's capsule, and flows through the renal tubule. Therenal tubule comprises four parts, the proximal convoluted tubule, theloop of Henle, the distal convoluted tubule and the collecting tubule.

The renal corpuscle comprises a tangled cluster of blood capillariescalled a glomerulus which is about 200 microns in diameter surrounded bya thin walled saclike structure called a glomerular capsule. Bloodenters and exits the glomerulus through the afferent and the efferentarteriole. While in the glomerulus, blood pressure causes water andvarious dissolved substances to be filtered out to the glomerularcapillaries into the glomerular capsule as glomerular filtrate.

The relative concentration of some of the substances in plasma,glomerular filtrate and urine is shown in Table I and Table II. Thesevalues may vary depending on many factors such as fluid consumption,medication, age, diet, health and kidney function of the patient.

TABLE I Glomerular Plasma Filtrate Urine Substance (mEq/l) (mEq/l)(mEq/l) Sodium 142 142 128 Potassium 5 5 60 Calcium 4 4 5 Magnesium 3 315 Chlorine 103 103 134 Bicarbonate 27 27 14 Sulfate 1 1 33 Phosphate 22 40

TABLE II Plasma Glomerular Filtrate Urine Substance (mg/100 ml) (mg/100ml) (mg/100 ml) Glucose 100 100 0 Urea 26 26 1820 Uric Acid 4 4 53Creatinine 1 1 196

The total rate of glomerular filtration typically is about 180 litersper day per person. Most of this volume is returned to the bloodstreamvia the process of reabsorption. Reabsorption is the movement ofsubstances out of the renal tubules into the blood. Substancesreabsorbed comprise water, glucose and other nutrients, sodium and otherions. Reabsorption begins in the proximal convoluted tubules andcontinues in the loop of Henle, distal convoluted tubules and collectingtubules.

Secretion is the process by which substances and fluids move into thedistal and collecting tubules from blood in the capillaries around thesetubules. Substances secreted are hydrogen ions, potassium ions, ammonia,and certain drugs. Kidney tubule secretion plays a crucial role inmaintaining the body's acid/base balance.

Homeostasis is maintained by the body by specialized hormones whichaffect the functions of the kidneys. The pituitary hormone ADH(antidiuretic hormone) decreases the amount of urine produced by makingdistal and collecting tubules permeable to water. Aldosterone, secretedby the adrenal gland controls the kidney tubules reabsorption of saltand other electrolytes. Primarily, aldosterone stimulates the tubules toreabsorb sodium at a faster rate.

While hormones affect kidney function, the kidneys also produce hormonesto regulate the function of other organs. Erythropoietin is a hormonesecreted by the kidney cells to regulate the rate of red blood cellformation. Renin, a second hormone secreted by the kidneys regulatesblood pressure. In addition, the kidneys activate vitamin D, which isinvolved in skeletal integrity.

To summarize, blood is treated and urine is formed as a result ofglomerular filtration of blood plasma, tubular reabsorption and tubularsecretion. In tubular reabsorption, substances such as glucose, aminoacids, proteins, creatine, lactic acid, citric acid, uric acid, ascorbicacid, phosphate ions, sulfate ion, calcium, potassium ions, sodium ionswater and urea are reabsorbed. In tubular secretion, penicillin,creatinine, histamine, phenobarbital, hydrogen ions, ammonia, andpotassium are secreted.

When both kidneys in a patient fail, the blood pressure may rise, fluidmay collect in the body, waste levels may build up to a harmful level inthe blood and red blood cell production may be reduced. When thishappens, treatment is needed to replace the function of the failedkidneys. Treatments for renal dysfunction include hemodialysis,peritoneal dialysis, and kidney transplants.

Hemodialysis is a treatment procedure that cleans and filters the bloodof a patient with renal inadequacy. The treatment procedure reduces thelevels of harmful wastes, extra salt and fluids. Hemodialysis also helpscontrol blood pressure and maintains the proper balance of chemicalssuch as potassium, sodium, and chloride in the body.

Hemodialysis uses a dialyzer, or special filter, to treat blood. Duringtreatment, blood from a patient travels through tubes into an externaldialyzer. The dialyzer filters out wastes and extra fluids and returnsthe newly cleaned blood into the body. A typical treatment regimen maycomprise three hemodialysis treatments per week for two to four hourseach time. During treatment, mobility is limited, but a patient canengage in activities which do not require excessive movements such asreading and writing.

The disadvantages of hemodialysis include side effects and complicationscaused by rapid changes in the patient's body fluid and chemical balanceduring treatment. Muscle cramps and hypotension are two common sideeffects. Hypotension, a sudden drop in blood pressure, may cause extremeweakness and dizziness.

It usually takes a few months for a patient to adjust to the sideeffects of hemodialysis. Side effects may be reduced by strict adherenceto the proper diet and the consumption of medicines as directed. Aproper diet helps to reduce the wastes that build up in a patient'sblood and reduces the load of the kidney. A dietitian is needed to helpplan meals according to a physician's instructions.

Further disadvantages of hemodialysis include high cost and frequent andlengthy travel to a dialysis center. An alternative to dialysis centersis home dialysis. A helper is required for home dialysis and both thepatient and the helper require special training. In addition, space isrequired for storing the machine and supplies at home.

Peritoneal dialysis uses the patient's abdomen lining, the peritonealmembrane, to filter blood. A cleansing solution, called dialysate,travels through a special tube into the patient's abdomen. Fluid,wastes, and chemicals pass from tiny blood vessels in the peritonealmembrane into the dialysate. After several hours, the dialysate isdrained from the abdomen, taking the wastes from the blood with it. Theabdomen is then filled with fresh dialysate and the cleaning processbegins again.

The dialysis procedure involves various degrees of difficulties andsignificant treatment times. While treatment regimens vary, theygenerally pose significant inconveniences. Typical treatment regimen maycomprise, for example, thirty to forty minutes every four to six hours,ten to twelve hours every night, thirty-six to forty-two hours per week,or 24 hour treatment sessions. In addition, special reduced calorie,potassium restricted diets are required in addition to dialysis.

Possible complications of peritoneal dialysis include peritonitis, orinfection of the peritoneum. The procedure of peritoneal dialysiscomprises many steps where pathogens such as bacteria may be introducedinto the body. Symptoms of peritonitis include inflammation, exudationsof serum fibrin cells and pus, nausea, dizziness, fever, abdominal pain,tenderness, constipation and vomiting. To avoid peritonitis, care isneeded to follow the procedure exactly. A patient needs to be trained torecognize the early signs of peritonitis. Failure to intervene quicklymay lead to serious problems.

In addition to short term inconveniences and side effects, hemodialysisand peritoneal dialysis have serious long term complications.Complications such as bone disease, high blood pressure, nerve damage,and anemia may have devastating effects with time. As a result of thesecomplications, 60% of kidney dialysis patients are unemployed and 30%are disabled. Kidney dialysis patients generally have shorter life spansand five fold higher hospitalization when compared to the generalpopulation.

Kidney transplantation is a procedure that places a healthy kidney froma donor person into a patient's body. The implanted kidney augments orreplaces the blood filtering load of the patient's failing kidneys. Theimplanted kidney is placed between the upper thigh and abdomen. Theartery and vein of the new kidney are connected to an artery and vein ofthe patient, and blood flows through the new kidney and makes urine. Thepatient's own kidneys, which may still be partially functional, are notremoved, unless they are causing infection or high blood pressure.

Like dialysis, non-histocompatible transplantation is not a cure. Tissuerejection is a significant risk even with a good histocompatibilitymatch. Immunosuppressive regimens to prevent rejection, based on drugssuch as cyclosporine, remain the cornerstone of mostpost-transplantation care. However the pharmaceuticalimmunosuppressant's narrow therapeutic window between adequateimmunosuppression and toxicity, as determined by the significantintrapatient and interpatient pharmacokinetic and pharmacodynamicvariabilities, renders it difficult to discern effective, but minimallytoxic immunosuppressive drug levels. Thus, post-transplantation carestill incurs significant costs and risks.

Prolonged immunosuppressant consumption may cause side effects. The mostserious is a weakened immune system, making it easier for infections todevelop. Some drugs also cause weight gain, acne, facial hair,cataracts, extra stomach acid, hip disease, liver or kidney damage.Diets for transplant patients are less limiting than for dialysispatients, but a patient is still required to cut back on some foods.Sometimes even immunosuppressants cannot prevent rejection of thekidney. If rejection happens, patients will be required to employ someform of dialysis and possibly wait for another transplant.

The time it takes to locate a kidney donor varies. There are not enoughcadaver donors for every person who needs a transplant and this problemis especially acute in the case of kidney transplants. Another source ofkidneys are from living donors such as relatives and spouses.Transplants from genetically related living donors often function betterthan transplants from cadaver donors because of betterhistocompatibility.

Humes (U.S. Pat. No. 5,429,938) has reported a method for culturingkidney cells for in vitro tubulogenesis and ex vivo construction ofrenal tubules. In the method, kidney cells are cultured in the presenceof tumor growth factor β1, epidermal growth factor and all-transretinoic acid to form three-dimensional aggregates. Among thedisadvantages of the method are the requirements for administration ofgrowth factors and the lack of glomeruli formation. Administration ofgrowth factors to a patient may have unwanted side effects andcomplications. While Humes disclosed a method which may help regrowth ofdamaged kidney tissue, a method for construction and use of a prosthetickidney was not disclosed.

The growth of liver (Rozga et al., Hepatology 17, 258-65) and blood(Schwartz et al., Blood 78, 3155-61) cells constrained in semiporousmembrane structures has been reported. These organ structures are notsuitable for prosthetic kidney construction because they do not allowfor the collection and excretion of glomerular filtrate outside thebody.

Naughton and Naughton (U.S. Pat. No. 5,516,680) have reported athree-dimensional kidney cell and tissue culture system. Athree-dimensional structure of living stromal cells is laid down on topof a stromal support matrix. Kidney cells are layered on top of thisthree dimensional system and cultured.

Vacanti and Langer (WO 88/03785) have disclosed methods for culturingcells in a three-dimensional polymer-cell scaffold of biodegradablepolymer. Organ cells, cultured within the polymer-cell scaffold, areimplanted into a patient's body to form an artificial organ.

Overall, therefore, it is apparent that the known methods of kidneyculture contain inherent defects and flaws and place specificlimitations on the ability to use the culture as a prosthetic kidneybecause of the limitations of their design. While each of these methodshas attempted to address some of the problems encountered in theconstruction of prosthetic kidneys, none of the disclosed methodssuggests a method for the construction of an actual prosthetic kidneycapable of filtering blood, producing glomerular filtrate, secretion, orreabsorption.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides methods, andapparatus for the treatment of kidney dysfunction and failure.

One embodiment of the invention is directed to a prosthetic kidneycomprising at least one artificial renal unit (ARU). The artificialrenal unit comprises a porous membrane structure having an externalsurface defining an enclosed internal space having at least one effluentchannel, and the membrane structure further having attached to theexternal surface thereof and in fluid communication with the enclosedinternal space thereof a plurality of nephron analogs. Each of thenephron analogs comprises a renal tubule analog having vascularizationforming a glomeruli-like structure in at least one region of the renaltubule analog. The renal tubule analog comprises a three-dimensionalcell aggregate of kidney tubule cells, the aggregate containing a lumenin fluid communication with the internal space of the membranestructure, and wherein the kidney tubule cells in the aggregate exhibita brush border.

Another embodiment of the invention is directed to an artificial renalunit comprising a porous membrane structure having an external surfacedefining an enclosed internal space having at least one effluentchannel, and the membrane structure further having attached to theexternal surface thereof and in fluid communication with the enclosedinternal space thereof a plurality of nephron analogs. Each of thenephron analogs comprises a renal tubule analog having vascularizationforming a glomeruli-like structure in at least one region of the renaltubule analog. The renal tubule analog comprises a three-dimensionalcell aggregate of kidney tubule cells, the aggregate containing a lumenin fluid communication with the internal space of the membranestructure, and wherein the kidney tubule cells exhibit a brush border.

Another embodiment of the invention is directed to an artificial renalunit precursor suitable for implanting in a patient with need ofadditional renal function comprising a porous membrane structure havingan external surface defining an enclosed internal space and having atleast one effluent channel. The membrane structure further havingattached to the external surface thereof and in fluid communication withthe enclosed internal space thereof, a plurality of renal tubuleanalogs. The renal tubule analogs comprise a three-dimensional aggregateof kidney tubule cells, the aggregate containing a lumen in fluidcommunication with the internal space of the membrane structure, andwherein the kidney tubule cells in the aggregate exhibit a brush border.

Another embodiment of the invention is directed to a method for makingan artificial renal unit precursor suitable for implantation into apatient in need of additional renal function comprising the steps ofproviding a porous membrane structure having an external surfacedefining an enclosed internal space having at least one effluentchannel; contacting the external surface with a suspension of kidneytissue cells; and culturing the kidney cells on the external surface invitro to form a plurality of renal tubule analogs, the renal tubuleanalogs comprising a three-dimensional aggregate of kidney tubule cells,the aggregate containing a lumen in fluid communication with theenclosed space of the membrane structure, and wherein the kidney tubulecells in the aggregate exhibit a brush border.

Another embodiment of the invention is directed to a method for treatingkidney disease, or augmenting renal function, in a patient comprisingthe steps of implanting an artificial renal unit precursor describedabove into the patient in an area having native vascular supply;inducing the native vascular supply to form glomeruli-like structures atthe one region; and connecting the effluent channel from the membranestructure to the urinary system of the patient.

Another embodiment of the invention is directed to a porous membranestructure for a prosthetic kidney comprising, a semipermeable membraneof a biocompatible polymer with an external surface defining an internalspace, and wherein the membrane structure comprises a plurality ofhollow tubes in fluid communication with a header and an effluentchannel on the header allowing drainage of the internal space.

Another embodiment of the invention is directed to a method for making arenal tubule analog comprising the steps of isolating kidney tissue;dissociating the kidney tissue by enzymatic treatment to form a cellsuspension; culturing the kidney cell suspension in vitro; treating anenclosed porous membrane structure with extracellular matrix protein;culturing the kidney cells on the treated exterior surface of theenclosed porous membrane structure to form renal tubule analogs, whereinthe renal tubule analogs comprise three-dimensional cell aggregates ofkidney tubule cells, containing lumens within the interior of theaggregates; and wherein the tubule cells exhibit a brush border.

Other embodiments and advantages of the invention are set forth, inpart, in the description which follows and, in part, will be obviousfrom this description and may be learned from the practice of theinvention.

DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIGS. 1A-D depict various collapsible configurations for the artificialrenal unit (ARU) or the porous membrane structure.

FIGS. 2A-C depict various inflatable collapsible configurations for theartificial renal unit ARU or the porous membrane structure.

FIGS. 3A-J depict various shapes for an artificial renal unit or aporous membrane structure.

FIG. 4 depicts an artificial renal device composed of polycarbonatetubular membranes with 4 micron pore size connected to collecting ductsand a reservoir.

FIG. 5 depicts immunocytochemical staining of a section of artificialrenal unit with anti-osteopontin antibody.

FIG. 6 depicts uniform staining for fibronectin in the extracellularmatrix of newly formed tubules.

FIGS. 7A-B depict sections from an ARU stained with anti-alkalinephosphatase antibody.

FIGS. 8A-B depict a section from an implanted ARU showing the formationof glomeruli-like structures and highly organized tubule-like structuresof different sizes.

DESCRIPTION OF THE INVENTION

As embodied and broadly described herein, the present invention isdirected to a prosthetic kidney, to methods of making a prosthetickidney and to methods of treating kidney disease with the prosthetickidney.

Each of the numerous methods and devices that has been used to treatkidney disease has attempted to solve one or more problems recognized asimportant for successful augmentation of kidney function. Examples ofthese methods include blood dialysis and various forms of peritonealdialysis. However, all these methods and devices have suffered fromproblems related to system complexity, large size and weight, longtreatment time or difficulties in controlling infections. The problemsassociated with current devices and methods of treating kidney diseasessuch as dialysis machines and intraperitoneal dialysis, include theinability to provide a high quality of life with high mobility, adequatekidney function for moderate exercise, freedom from a strict dietaryregimen, and freedom from complications such as, for example, infection,nausea, pruritus, poor nutrition, pseudo gout, hepatitis B infection,accelerated cardiovascular disease, hypertension, renal osteodystrophy,anemia, pleural effusion, thrombosis, serositis, pericarditis, dialysisascites, and dialysis dementia.

It has been demonstrated that transplanted kidneys may be effectivelyused on a widespread basis to treat kidney disease and that long-termsurvival can be obtained. However, to date, there are insufficient donorkidneys available for all patients who require one and no artificialkidney has been developed which is truly practical for widespreadapplication. Additionally, present kidney transplant patients require avery extensive level of follow up care, including medical management ofthe patient's immune system.

An object of the present invention is to provide a prosthetic kidneycapable of sustaining a high quality of life for many years, with a lowrisk of complications associated with current methods of treating kidneydisease such as, vomiting, infection, hypotension, cramps, bleeding,leukopenia, hypoxia, electrolyte disturbances, and dialysisdisequilibrium and nausea.

Another object of the invention is to provide a prosthetic kidney thatfunctions effectively at high filtration rates and, therefore, occupiesa reasonably small volume.

Another object of the invention is to provide prosthetic kidneys thatcan be implanted within the patient to replace or augment the functionof the natural kidneys.

Another object of the invention is to provide a method of makingprosthetic kidneys from a patient's own kidney cells or from donorcells.

Another object of the invention is to provide prosthetic kidneys thatare inherently histocompatible, durable and reliable and are capable offiltrating blood for extended periods.

Another object of the invention is to provide a prosthetic kidney withredundancy to allow adequate renal function in the event of failure ofone or more of its components.

Another object of the invention is to provide a prosthetic kidneycomprising a biodegradable structure which may fill an initialstructural role for the formation of the prosthetic kidney and thenpromote tissue growth as the biodegradable structure degrades.

Another object of the invention is to provide a prosthetic kidney withan effluent drainage and collection system.

One embodiment of the invention is directed to a prosthetic kidneycomprising one or more artificial renal units (ARU). One preferredprocess for making such a device will now be described.

Harvesting Kidney Cells

The ARU is constructed in part using kidney cells from a donor. In anautologous prosthetic kidney, the kidney cells may be derived from thepatient's own kidneys. In an allogeneic prosthetic kidney, the kidneycells may be derived from other member of the patient's species. In axenogenic prosthetic kidney, the kidney cells may be derived from aspecies different from the patient. Donor cells may be from the cortexof the kidney of many mammalian sources such as, for example, humans,bovine, porcine, equine, caprine and ovine sources. Kidney cells may beisolated in biopsies, or autopsies. In addition, the cells may be frozenor expanded before use.

To prepare for ARU construction, a kidney or a kidney tissue section isdissociated into a cell suspension (Example 1). Dissociation of thecells to the single cell stage is not essential for the initial primaryculture because single cell suspension may be reached after a period,such as, a week, of in vitro culture. Tissue dissociation may beperformed by mechanical and enzymatic disruption of the extracellularmatrix and the intercellular junctions that hold the cells together.Kidney cells from all developmental stages, such as, fetal, neonatal,juvenile to adult may be used.

Higher yields of cells may be obtained from fetal or neonatal tissue. Toreduce stress on the cell and to facilitate handling, a section ofkidney tissue may be placed in an balanced salt solution beforedisruption. Examples of balanced salt solutions include, for example,tissue culture medium, Hank's balanced salt solution and phosphatebuffered saline and the like which are available from commercial sourcessuch as Gibco (Gaithersburg, Md.) and Sigma (St. Louis, Mo.). The kidneytissue may be disrupted by mechanical tissue disrupter such as ascalpel, needles, scissors or a cell dissociation sieve (Sigma, St.Louis, Mo.). After mechanical disruption, the tissue may be furthertreated enzymatically with proteolytic enzymes such as trypsin,collagenase, dispase, and with agents that bind or chelate calcium ionsuch as ethylenediaminetetraacetic acid (EDTA). Calcium chelators removethe bioavailability of calcium ions on which cell-to-cell adhesiondepends.

To maintain cell viability, enzymatic dissociation may be monitored byeye or by microscope. The enzymes may be inactivated after achievingdesired dissociation but before cell viability is substantiallyimpaired. Enzyme inactivation may be performed by isotonic washing, orserum treatment.

Primary cultures may be prepared from the dissociated cells with orwithout a cell fractionation step. Cell fractionation may be performedusing techniques, such as florescent activated cell sorting, which isknown to those of skill in the art. Cell fractionation may be performedbased on cell size, DNA content, cell surface antigens, and viability.For example, tubule cells may be enriched and fibroblast cells may bereduced. While cell fractionation may be used, it is not necessary forthe practice of the invention.

Cell sorting may be desirable, for example, when the donor has diseasessuch as kidney cancer or metastasis of other tumors to the kidney. Akidney cell population may be sorted to separate malignant kidney cellsor other tumor cells from normal non-cancerous kidney cells. The normalnon-cancerous kidney cells, isolated from one or more sortingtechniques, may be used to produce a prosthetic kidney.

Another optional procedure in the method is cryopreservation. Cryogenicpreservation may be useful, for example, to reduce the need for multipleinvasive surgical procedures. Cells taken from a kidney may be amplifiedand a portion of the amplified cells may be used and another portion maybe cryogenically preserved. The ability to amplify and preserve cellsallow considerable flexibility in the choice of donor cells. Forexample, cells from a histocompatible donor, may be amplified and usedin more than one recipient. An added advantage with cryopreservation andamplification is that cells from one kidney may be amplified to producea second kidney.

Another example of the utility of cryogenic preservation is in tissuebanks. Donor cells may be cryopreserved along with histocompatibilitydata Donor cells may be stored, for example, in a donor tissue bank. Astissue is needed to treat kidney disease in a patient, cells may beselected which are most histocompatible to the patient. Patients whohave a disease or undergoing treatment which may endanger their kidneysmay cryogenically preserve a biopsy of their kidneys. Later, if thepatient's own kidneys fail, the cryogenically preserved kidney cells maybe thawed and used for treatment. Examples of diseases which damageskidneys include, for example, high blood pressure and diabetes. Examplesof kidney-damaging treatment procedures include, for example,chemotherapy and radiation.

Culturing Kidney Cells

It may be desirable to amplify the number of cells to produce sufficientstarting material for the ARU. Amplification offers advantages such asreduction of the amount of donor tissue needed. For example, a smallkidney biopsy section may be removed from a living donor. The removedtissue may be sufficiently small such that the donor's renal functionand capacity is not substantially reduced. The removed kidney biopsysection may be amplified to provide a prosthetic kidney for one or morepatients. In some situations, such as an autologous transplant where thepatient has already suffered substantial kidney damage, the ability toamplify kidney cells and regenerate ARUs has substantial benefits.

Cell amplification may be accomplished by repeatedly subculturing thecells into successively larger or more numerous culture vessels. Cellsmay be repeatedly subcultured for weeks, months and years. Thus, onesmall section of kidney, for example, less than one gram of tissue froma biopsy, may after repeated culturing, be expanded into about 10 grams,about 50 grams, about 200 grams or more of tissue for use in theconstruction of ARUs.

In one preferred method, cells are cultured and amplified underconditions discussed in Example 2 and 3. Briefly, all cells are culturedon collagen treated plates, in Dulbecco's Modified Eagles's Mediumsupplemented with about 10% fetal bovine serum, about 5 μg/ml bovineinsulin, about 10 μg/ml transferrin, about 10 μg/ml sodium selenite,about 0.5 μM hydrocortisone, about 10 ng/ml prostaglandin E₂, about 100units/ml penicillin G, about 100 μg/ml streptomycin in an incubator atabout 37° C. and about 5% CO₂. All media and reagents may be purchasedcommercially from tissue culture supply sources such as, for example,Sigma of St. Louis, Mo.

Other culture media, such as, for example, MCDB, 199, CMRL, RPMI, F10,F-12, MEM, and the like are available from commercial sources such asGibco (Gaithersburg, Md.) and Sigma (St. Louis, Mo.) may also be used toculture kidney cells. Supplements may also be supplied by increasedserum concentration such as, for example, about 15%, about 20%, about30%, about 40% or about 50% serum. Compositions of various suitablemammalian cell media, serum, balanced salt solutions, and supplementsare listed in the Gibco (Gaithersburg, Md.) and Sigma (St. Louis, Mo.)catalogs which is hereby incorporated by reference.

The culture medium and matrix used for the maintenance of viable kidneycells over long periods should be chosen in order to keep these cells ina hormonally responsive state. Small amounts of insulin, hydrocortisoneand retinoic acid are desirable to maintain cells capable of formingrenal tubule analogs and artificial renal units. Insulin, hydrocortisoneand retinoic acid are especially desirable especially at longerculturing periods encountered during amplification of cells.

Porous Membrane Structure

The ARU of the present invention includes an enclosed porous membranestructure. This porous membrane structure comprises a porous membranewith an exterior surface defining an enclosed interior space and atleast one effluent channel. As described below, renal tubule analogs areformed on the exterior surface of the porous membrane structure. In themost simple embodiment of the invention, the membrane structure maycomprise a hollow tube with both ends sealed. The effluent channel maybe attached to the enclosed tube to allow drainage of the interiorspace.

A porous membrane structure of the invention also may be, for example, aplurality of hollow porous tubes or hollow fibers, each closed at oneend and in fluid communication with a header at the other end. Hollowfibers may be purchased commercially and the construction of hollowfiber for cell culture is well known to those in the art. One method ofhollow fiber fabrication involves the extrusion of membrane polymersthrough fine dies to form tubes which harden and form tubes which arehighly porous. Several hundred to several thousand to tens of thousandsof fibers maybe connected to a header. Further, multiple headers may beconnected by fluid channels to form a superstructure for the prosthetickidney. Hollow fiber construction can pack a large surface to volumeratio into a relatively small volume.

Alternate porous membrane structures may comprise a plurality of hollowporous tubes or fibers open in both ends and in fluid communication atone end with a first header and in fluid communication at a second endwith a second header. This design may allow flushing, or the passage ofa sweeping fluid, through the porous membrane structure. Flushing mayallow cleaning, disinfection, treatment, or delivery of drugs orchemicals before use and during use of the porous membrane structure.

Porous membrane structures may also be constructed by sintering-fusionof particles to form a three-dimensional structure. Other methods forthe construction of porous membrane structures include casing,stretching, leaching, nucleation and laser fabrication. In casing, athin film of solution containing the polymer and solvent is cast,sometimes on a base of cloth or paper. Solvent escapes and polymerprecipitation cause pore formation. In stretching, Teflon™,polypropylene or other polymer sheets are stretched equally in alldirections creating uniform pores. In leaching, a solution containingtwo materials is spread as a film. Next a solvent is used to dissolveaway one of the components, resulting in pore formation. (See Mikos,U.S. Pat. No. 5,514,378, hereby incorporated by reference) Innucleation, thin polycarbonate films are exposed to radioactive fissionproducts that create tracks of radiation damaged material. Next thepolycarbonate sheets are etched with acid or base, turning the tracks ofradiation-damaged material into pores. Finally, a laser may be used toburn individual holes through many materials to form porous membranestructures. In addition to polymeric material, metals, glasses, andceramics may also be used to form porous membrane structure with laserfabrication. An electronic microscope may be used for quality controland to determine the structure of the membranes following any method offabrication.

The porous membrane structures may be fabricated with natural orsynthetic polymeric biocompatible materials such as cellulose ether,cellulose, cellulosic ester, fluorinated polyethylene, phenolic,poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide,polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether,polyester, polyestercarbonate, polyether, polyetheretherketone,polyetherimide, polyetherketone, polyethersulfone, polyethylene,polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenyleneoxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide,polysulfone, polytetrafluoroethylene, polythioether, polytriazole,polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose,silicone, urea-formaldehyde, or copolymers or physical blends thereof.The polymer material should be selected to be compatible with, andresistant to attack by body fluids and host cells. Further, it ispreferred that the material is resistant to attack by any drugs orchemicals, such as medicine or chemotherapy material which the patientmay be subjected as part of the treatment of kidney disease or any otherdisease the patient may also have.

The porous membrane material may be biodegradable and designed to slowlydegrade in a body. Biodegradable materials may fill an initialstructural role for the formation of the prosthetic kidney and thenpromote tissue growth as they degrade. Further, it is preferred that thebiodegradable material and its degraded product are non-toxic and can beeasily eliminated from the system of a disease patient or a healthypatient. Representative materials for forming the biodegradablestructure include natural or synthetic polymers, such as, for example,poly(alpha esters) such as poly(lactate acid), poly(glycolic acid),polyorthoesters and polyanhydrides and their copolymers, which degradedby hydrolysis at a controlled rate and are reabsorbed. These materialsprovide the maximum control of degradability, manageability, size andconfiguration.

The pores on the porous membrane structure should be sufficiently largeto allow passage for fluids and gas but sufficiently small to beimpermeable to cells. Suitable pore size to accomplish this objectivemay be about 0.04 micron to about 10 microns in diameter, preferablybetween about 0.4 micron to about 4 microns in diameter. Polycarbonatemembranes are especially suitable because they can be fabricated in verycontrolled pore sizes such as, for example, about 0.01 microns, about0.05 micron, about 0.1 micron, about 0.2 micron, about 0.45 micron,about 0.6 micron, about 1.0 micron, about 2.0 microns and about 4.0microns. At the submicron level the porous membrane may be impermeableto bacteria, viruses and other microbes.

The porous membrane structure may be completely porous or partiallyporous. It may be desirable for regions of the porous membrane structureto be non-porous. Non-porous regions may include, for example, hingedregions, structural regions, effluent and affluent ports, connectors andattachment sites.

The effluent channel may be any outlet opening in the porous membranestructure such as, for example, nozzle, tube, fitting, hole, or openingor the like wherein the fluid may exit from the interior space of theporous membrane structure. Optionally, a non-refluxing valve, commonlyknow as a check valve, may be incorporated into the effluent channel toprevent reflux of effluent back into the porous membrane structure. Afilter, with pores sufficiently large to allow passage of fluids butsufficiently small to prevent passage of microbes may be added to theeffluent channel to prevent or reduce the possibility of infection ofthe prosthetic kidney. Optionally, means for collection and storage ofeffluent such as an artificial bladder, a chamber, a tube or the likemay be connected to the effluent channel or the porous membranestructure. Effluent may be stored between periods of drainage or forsubsequent analysis.

The porous membrane structure may be a collapsible structure such as,for example, the structures shown in FIG. 1. A collapsible structure isany structure that can be deployed in one shape and retracted into asecond more compact shape. FIG. 1 include examples of structures whichcan transform shapes, such as, for example, from planar to bulbous (FIG.1A), from elongated to helical (FIG. 1B), from elongated to superhelical(FIG. 1C) and from cylindric to bellow structures (FIG. 1D). Thedeployed shape may be more suitable for seeding of kidney cells or forimplantation which the retracted shape may be more suitable for longterm implants. The deployed shape of the collapsible porous membranestructure may be, for example, a long or flat cylinder (FIGS. 3A, 3G),an elongated tube (FIGS. 3B, 3E), a coil (FIG. 3K), a helix (FIGS. 1Band 3C), a double or multiple helix (FIG. 1C), a kidney shape (FIG. 3D),a cube (FIG. 3H), a flat bag (FIG. 3I), or combination thereof Theporous membrane structure may further comprise bellows 10 (FIG. 1D),tracts, strings or wire 20 (FIG. 1D) for the pre-implantation orpost-implantation deformation and shaping of the structure. Further theporous membrane structure may comprise support to maintain integrity andto prevent pressure necrosis of the cells of the ARU. Such support maycomprise pillars, ribs or the like which may be attached or not attachedand positioned internal or external to the porous membrane structure.The retracted shape of the collapsible porous membrane structure may besimilar to the deployed shape except more complex. Other retractedshapes may comprise planes, spheres and kidney shapes. Other desirablecollapsible structures may comprise retracted shapes that are designedto fit subcutaneously or within the abdomen or thorax of a patient.

Examples of collapsible structure suitable for prosthetic kidney includean accordion-like or bellows type collapsible structure (FIG. 2C), mostpreferably formed of a porous biocompatible polymer. Foldable orcollapsible porous membrane structures can be deployed during theattachment of the kidney cells. Then the collapsible structure, in thedeployed form may be implanted into a patient. As neo-angiogenicactivity of the patient perfuse the prosthetic kidney, the structure maybe slowly retracted. Alternatively, after attachment of kidney cells,the structure may be retracted and implanted into a patient. Collapsiblethree-dimensional porous membrane structures can be developed in avariety of forms. It is preferred that the three-dimensional collapsiblemembrane structure have a desirable final and initial geometry.Desirable geometry is dictated by the location of implantation of theprosthetic kidney. A prosthetic kidney designed to be implantedsubcutaneously may be of a planar shape while a prosthetic kidney forthe replacement of a normal kidney may be of a kidney shape. Anotherdesign consideration is that the structure must be deployable withoutcausing disintegrating or component failure either in the collapsed ordeployed state. For example, it is desirable for such a structure tomaintain structural integrity and not to have any occlusion in theexpanded and collapsed state which may cut off or reduce blood supply tothe prosthetic kidney.

Another method for forming a collapsible porous membrane structure is aninflatable structure (FIGS. 2A, 2B, and 2C). For example one embodimentof an inflatable collapsible structure and its corresponding crosssectional structure is shown in FIGS. 2A and 2B respectively. Thestructure may comprise an inflatable body 10 having a flexible butsubstantially non-elastic and transversely convex wall 20. Thetransversely convex walls may be elongated and adapted to be bowed orconcave and hence flexible through its width. The inflatable structuremay be inflated for the seeding of kidney cells and deflated eitherbefore or after implantation. Another embodiment of an inflatablecollapsible porous membrane structure is accordion shaped and shown inFIG. 2C.

The enclosed porous membrane structure may optionally comprise adeformable outer casing with sufficient strength to prevent mechanicaldamage to the ARU. The deformable casing may be shaped into any numberof desirable configurations to satisfy any number of overall system,geometry or space restrictions. The deformable casing be formed fromfilm, gauze, webbing, wire mesh, cloth, foam rubber and the like.Suitable materials for the construction of the casing include, forexample, metals such as steel, titanium, glass, polymers, fiberglass,plastic or the like. The final shape of the porous membrane structurewith the outer casing may be, for example, a cylinder (FIGS. 3A, 3B,3G), a coil (FIG. 3J), a helix (FIG. 3C), a double or multiple helix(FIG. 1C), a kidney shape (FIG. 3D), a cube (FIG. 3H), a flat bag (FIG.3I) or combination thereof (FIG. 4). The only limitation on degree ofdeformation is that a significant area of the porous membrane structureand its associated ARUs not be significantly broken, stretched, crimped,or put under excessive stress, pressure, or compression such that therenal function of the prosthetic kidney is impaired, diminished orotherwise deleteriously affected.

In an embodiment of the invention, the porous membrane structure may bemade with materials which are radiation resistant such that radiationtherapy will not unfavorably alter the renal functions of the prosthetickidney. In the treatment of certain diseases, such as for example,cancer of the kidneys, it may be necessary to apply radiationpostoperatively. The radiation resistant porous membrane structure wouldpermit heavy radiation to be applied with prosthetic kidney in place forresidual tumor treatment if necessary.

In an embodiment of the invention, the porous membrane structure is madewith materials which are ultrasound resistant. Calculi buildup andencrustation, such as for example, kidney stones, in the urinary systemis a common problem among patients with reduced renal abilities. Onemethod to treat calculi buildup, encrustations, and stones is to useultrasonic energy to crush the buildup. An ultrasound resistant porousmembrane structure will allow post-implantation ultrasound treatmentwithout unfavorably altering the prosthetic kidney.

Another embodiment of the invention is directed to a means fordelivering a drug, growth hormone and a means for controlling calculibuildup and encrustation in the porous membrane structure of theartificial renal unit (ARU) by providing at least one affluent channelto the porous membrane structure. Affluent channels may be any meanswhich allow an introduction of solid, liquids or gas into the porousmembrane structure such as a port or a fitting. Affluent channels may bepositioned at a point distal from the effluent channels to allow flow ofintroduced fluids to pass over substantially the entire interior surfaceof the porous membrane structure before exiting by the effluent channel.

It is known to those of skill in the art that combinations of growthfactors, such as transforming growth factor-β₁, epidermal growth factor,and all-trans retinoic acid may induce kidney tubule formation. It isalso known that various chemicals and pharmaceuticals such as potassiumcitrate and acetohydroxamic acid inhibit crystallization of poorlysoluble calcium and magnesium salts. It is known that various chemicalsand pharmaceuticals such as antibiotic and metallic compounds mayinhibit the growth of, or kill, microbes. Growth factors,anti-encrustation factors, and antibiotics may be added to the porousmembrane structure, in liquid form, by the affluent channel. Theaffluent channel may further comprise for example a tube which extendsfrom the ARU to a subcutaneous location. For example, in a patient withan abdominal ARU, the addition of growth factors, anti-encrustationfactors, and antibiotics may easily be accomplished by connecting to asubcutaneously implanted inlet port accessed by a hypodermic needle.

One embodiment of the invention provides a coating of extracellularmatrix material on the porous membrane structure for the growth anddevelopment of renal tubule analogs in vitro, The rapidthree-dimensional growth of kidney cells for construction of ARUs may befacilitated by the availability of biomaterial matrices upon which cellscan reside and regenerate. Thus, it may be preferable to treat theexterior surface of the porous membrane structure with materials thatgive appropriate signals for cellular growth control, such asextracellular matrix proteins.

Extracellular matrix proteins comprise glycoproteins, proteoglycans andcollagen. The exterior surface of the porous membrane structure may betreated with one or a combination of these proteins. Examples ofsuitable extracellular proteins which may be used include collagen,fibronectin, thrombospondin, cytotactin, echinonectin, entactin,laminin, tenascin, uvomorulin, vitronectin, biglycan, chondroitinsulfate, decorin, dermatan sulfate, heparin, heparin sulfate andhyaluronic acid.

The extracellular matrix protein may be extracted freshly from amammalian source such as rat tail. Other mammalian sources of collageninclude bovine, porcine and human sources. Human extracellular proteinsuch as collagen may be collected from human tissues such as placentaeor cadavers and may be purchased from commercial suppliers such as Sigma(St. Louis, Mo.).

Extracellular matrix protein and the porous membrane structure may besterilized prior to use. Sterilization may reduce the likelihood ofmicroorganism contamination of the extracellular matrix protein.Preferred methods for sterilizing include ultrafiltration and radiationexposure. Radiation exposure may be, for example, gamma radiation orX-radiation exposure. Alternatively, antibiotics, antibacterials andcytotoxic agents, ultra violet radiation, in normally effective dosesmay be used. One preferred cytotoxic agent used is ethylene oxide.

Surface treatment of the porous membrane structure may comprisecontacting a solution of ECM protein with the porous membrane structure.After contact, the pH may be raised, for example, by ammonium hydroxideto promote attachment, gelling, or polymerization. Alternatively, theextracelluar matrix protein may be crosslinked to the porous membranestructure. Crosslinking and derivatization reagents may be purchasedfrom a commercial supplier such as Pierce (Rockford, Ill.).

Seeding Kidney Cells Onto the Porous Membrane Structure

Attachment of the kidney cells to the exterior surface of the porousmembrane structure may be accomplished by combining the dissociatedkidney cells with the porous membrane structure. One preferred method isdescribed in detail in Example 3. Briefly, a porous membrane structureis gently contacted with a suspension of kidney cells at a density ofabout 1×10⁷ cells per square centimeter of porous membrane structuresurface. The coated porous membrane structure is incubated in a tissueculture incubator under about 100% humidity, about 37° C., about 5% CO₂for about 30 minutes to about one hour. After this period, medium isgently added to completely submerge the porous membrane structure.

The extracellular membrane protein on the porous membrane structure maypromote attachment of kidney cells and this attachment is substantiallycomplete within about 30 minutes to about 24 hours such as, for example,within 1 hour, 2 hours, 4 hours, 8 hours or 16 hours. The exact timerequired for complete attachment depends on the surface property andsurface composition of the porous membrane structure, the medium, andthe extracellular matrix and the kidney cells.

Culturing the Kidney Cells on the Porous Membrane Structure to FormRenal Tubule Analogs

After attachment, the structure containing kidney cells is cultured invitro for a time, and under conditions sufficient to form renal tubuleanalogs. For example, culture under about 5% CO₂ and about 37° C. forabout 3 days to about 20 days, preferably between about 7 to about 10days is generally sufficient. The medium covering the porous membranestructure may be replaced as required in time intervals of about 1 dayto about 6 days, preferably between 2 days and 5 days, more preferablybetween 3 days and 4 days. The interval of media replacement depends onthe volume of media and the rate of consumption of the nutrients in themedia and the rate of accumulation of waste products. It is understoodthat by adjusting the volume of media, longer period such as 7 days, 8days, 9 days or 10 days may elapse between media changes. It isunderstood that the addition of nutrients, antibiotics and hormones suchas fetal bovine serum, insulin, transferrin, sodium selenite,hydrocortisone, prostaglandin E₂, penicillin, and streptomycin mayextend the period between media changes.

Extracellular matrix protein treatment alters the surface properties ofthe porous membrane structure to affect attached cell morphology andmigration. When cultured on the treated porous membrane structure,kidney cells initially spread out as a monolayer, but over timespontaneously self-assemble into three-dimensional cell aggregates eachcomprising an interior lumen. The shape of the cell aggregates resemblesa linear segment of a renal tubule. Cell aggregates are attached to thesubstrate and have a smooth surface with practically indistinguishablecell-cell boundaries. The renal tubule analogs may be open or closed atone end. Renal tubule analogs on a per cell basis, show higher kidneyspecific activities compared to the kidney cell monolayers and remainviable longer. Specific cellular functions of tubule cells includesecretion, absorption, and the expression of cell specific gene productssuch as alkaline phosphatase (FIGS. 7A and B) and osteopontin (FIG. 5).Renal tubule analogs are attached to the porous membrane structure (FIG.7B) and exhibit a more tissue like ultrastructure than renal cells in amonolayer.

The evolution of kidney specific activity over the course of a renaltubule analog self-assembly on the porous membrane structure may bemonitored phenotypically and functionally. Phenotypically, renal tubuleanalogs have characteristics, such as brush border, lumens, and celljunctions similar to natural tubules. Expression of kidney tubulespecific genes such as osteopontin and alkaline phosphatase may bemonitored by any molecular biology or immunology methods known to thoseof skill in the art such as northern blots, western blots, polymerasechain protection and in situ hybridizations. Tests for activitieslocalized to kidney proximal tubule cells such as, for example, PeriodicAcid Schiff (PAS) test for glucose metabolism may be used to detectkidney proximal phenotypes.

Kidney proximal tubules were observed to form continuous sheets of ovalto elongated cells on the surface the porous membrane structure.Initially, 24 hours after seeding, the kidney cells on the porousmembrane structure are one cell layer thick but as they approachconfluence, regions may be found in which several cell layers arepresent. Each cell of the renal tubule analog has a prominent roundnucleus with one or two distinct nucleoli. Intracellular spaces arelarge with cells being attached at regions which resemble intracellularbridges. In some regions distinct infolding or pockets into the cellularsurface are in evidence. Some cells contain large number of granules,presumably lysosomes, characteristic of proximal tubule cells. Thisgranularity of the cells increased significantly in cultures which weremore than 7 days old.

When examined under the microscope, renal tubule analogs, like naturaltubules, show large intracellular spaces filled with long slendermicrovillar projections from the cell surface which resembles a brushborder. Brush borders, also called striated borders are uniquelycharacteristic of the apical surface of the proximal kidney tubulecells. In the microscope, brush borders appear as a specialization ofthe free surface of a cell, consisting of minute cylindrical processes(microvilli) that greatly increase the surface area. The brush border onrenal tubule analogs shows an extensive microvillar surface which iscontinuous into the infolding or pockets which form along the cellsurface. Histology sections cut through the infolding, parallel to butbelow the cell surface, appear as microvillar lined channels surroundedby cytoplasm. The formation of brush border or microvilli lined pocketsmay represent the mechanism by which a highly microvillous cuboidal cellmaintains its extensive apical surface area in an otherwise squamouscell culture environment. In regions of the cell surface lackingmicrovilli linked pockets or channels, the borders of cells also havelong microvilli projecting from them. However, their number is reducedand their structural organization is different from the brush borderobserved lining the pockets or channels and adjacent surfaces. Theregions of lower microvillar numbers may be the lateral and basalsurfaces of normally cuboidal kidney tubule cells.

The cells of the renal tubule analogs contain the intracellularorganizations typical of intact proximal tubule cells. The nuclei of thetubule cells are oval and contain scattered heterochromatin primarily atthe peripherae of the nucleus. Much of the nuclear material iseuchromatic in appearance and one or two nucleoli are visible uponmicroscopic examination. The cytoplasm of the cells of the renal tubuleanalogs contains numerous filamentous mitochondria often arrangedparallel to the surface of the cells. The shelf like cristae aregenerally arranged perpendicular to the length of the mitochondrion. Inthe more elongated cells, fine cytoplasmic microfilament bundles andextensive networks of cytoplasmic microfilaments are also presentespecially in continuity with intracellular junctions. These filamentousbundles run parallel to the lateral surface of the cells. In most cellsshort profiles of granular endoplasmic reticulums are present althoughin some cells they may become very extensive. Other cellular organellesinclude dense lysosomal granules and prominent Golgi networks. Lysosomecontent may increase significantly in older culture.

Individual desmosomes can be found at sites of contact along the lateralmargins of adjacent cells. Where groups of the more elongated cells makecontact in a single region, very elaborate desmosome-like junctionalcomplexes may form resembling the intercalated discs of cardiac muscle.Radiating from these complexes are extensive networks of monofilaments.These junctional complexes resemble the belt desmosomes of the apicalborder of normal proximal tubule cells. Thus, it is evident that on thebasis of their morphology, the renal tubule analogs have all thecharacteristics of proximal kidney tubules.

Gene expression analysis also indicates that the renal tubule analogsexpress genes typical of natural tubules. Natural kidney tubules expressosteopontin throughout the length of the renal tubule and expressalkaline phosphatase preferentially at the proximal end. Kidney cells,as a monolayer culture exhibit very low levels of osteopontin andalkaline phosphatase activity which can be seen by the low level ofstaining in sections probed with anti-osteopontin and anti-alkalinephosphatase antibodies. As the kidney cells begin to aggregate intorenal tubule analogs, the alkaline phosphatase activity increases.Within a single renal tubule analog, the distribution of this activityis heterogeneous. The proximal end of the renal tubule analog showsdetectable levels of alkaline phosphatase. If the cells are dissociated,such as, for example, by trypsin, the cells lose their enhanced activityand return to the low levels seen in the initial monolayers. Cells whichremain in a renal tubule analog structure, however, retain theirenhanced activity. It is hypothesized that their enhanced cell-cellcontact and more tissue-like structure contribute to the enhancedactivity seen in renal tubule analogs.

Implantation of Artificial Renal Unit Precursor

The Artificial Renal Unit Precursor, which comprises an enclosed porousmembrane structure with renal tubule analogs attached may be implantedinto a host to induce the formation of nephron analogs by glomeruliformation at the end of the tubules. Angiogenesis is important inprosthetic kidney function. The function and growth of a prosthetickidney require a blood supply.

In angiogenesis, the host tissue responds to signals produced by thecells of the prosthetic kidney. This response appears to include atleast three components. First, the capillary endothelial cells of therenal tubule analog breach the basal lamina that surrounds an existingblood vessel; endothelial cells of the host during angiogenesis havebeen shown to secrete proteases, such as plasminogen activator, whichenable them to digest their way through the basal lamina of the parentcapillary or venule. Second, the host endothelial cells migrate towardthe kidney cells. Third, the endothelial cells proliferate and formcapillaries perfusing the glomeruli-like structure which forms at theproximal end of the renal tubule analogs. The resulting structure,termed a nephron analog is formed in vivo by implanting a renal tubuleanalog into a host and inducing glomeruli formation on the proximal endof the renal tubule analog. Glomeruli formation may be seen on the endsof some of the renal tubule analogs by two weeks post-implantation. Ateight weeks post-implantation, glomeruli formation is extensive andvisible on most renal tubule analogs.

The artificial renal unit precursor has the ability to induce theformation of neovasculature and may be implanted both in lessvascularized or highly vascularized regions of a patient's body. Oneweek after subcutaneous implantation, vascular formation is extensivealong the length of the renal tubule analogs. The renal tubule analogsdevelop into nephron analogs by the end of eight (8) weeks byangiogenesis along the tubule and glomeruli formation in at least oneregion of the renal tubule analog. A plurality of glomeruli or glomerulilike structures are also seen on each renal tubule analog. Histologicalobservations detected extensive vascularization along the length of eachrenal tubule analog.

The ARU examined show characteristics of proximal kidney tubules andglomeruli. ARUs express alkaline phosphatase and gamma glutamyltransferase, two genes which show almost exclusive expression inproximal tubule cells. Artificial glomeruli when tested functionally orimmunohistochemically, show the presence of coagulation factor VIII.

Because a prosthetic kidney without a blood supply relies on diffusionfor a supply of nutrients, a prosthetic kidney may be limited to aliving layer not thicker than a few millimeters until angiogenesis canprovide adequate perfusion. One embodiment of the invention is directedto a method to overcome this limitation by implanting multiple ARUstructures into a host and allowing angiogenesis to connect the ARUs toan artery or a vein. Then the ARUs, along with an artery and vein areremoved from the host and surgically combined into a larger or thickerARU and reimplanted into a patient. The artery and vein of the ARU areconnected to an artery and vein of the patient to provide a blood supplyto the assembled prosthetic kidney.

Connection of the Effluent Channel

The effluent channel of the ARU may be connected to any location in apatient to allow removal of effluent from the prosthetic kidney. In anembodiment of the invention, the effluent channel may be connected to apart in the patient to allow removal of filtrate from the prosthetickidney. The effluent channel may be connected to the urinary tract,including the kidney, the ureter, the renal pelvis, the bladder, theurethra, the testicle, prostate or vas differens. Alternatively, theeffluent channel may be connected to the intestine, such as a largeintestine or small intestine, in an intestinal conduit operation.Further, the effluent channel may be extended by a tube to protrudethrough the skin of the patient in whom the prosthetic kidney isimplanted. In an abdominal implant of a prosthetic kidney, for example,the effluent channel may be extended through the abdominal wall. A capmay be installed on the end of the effluent channel to prevent leakageof effluent. When desired, the patient may remove the cap and alloweffluent to be discharged externally.

The attachment of the effluent channel may be by sutures. In order thatthe ARU can be tailored by the surgeon installing the ARU in a patient,the effluent channel of different ARU may comprise different sizes sothey can be matched to the effluent channel attachment site. Further,the effluent channel may be constructed of a material which would allowsimple tailoring during an implant to fit the situation and location atthe time of the operation.

In Vitro Operation

In another embodiment of the invention, the prosthetic kidney may beremoved from the host along with its artery and vein and cultured invitro. A prosthetic kidney with nephron analogs may be used as a benchtop bioreactor to form a bench top prosthetic kidney. In vitroprosthetic kidneys may be fed using mammalian serums or by connectiondirectly to a mammal. An initial pilot scale in vitro prosthetic kidneymay be used as the starting material for an eventual production scale invitro prosthetic kidney. For example, additional porous membranestructures may be attached to the prosthetic kidney to induce it to growin size in culture. The in vitro prosthetic kidney may be eventuallyimplanted into a patient as an individualized therapeutic product. Theprosthetic kidney may be preserved and multiplied to such an extent invitro that large scale industrial processing and harvest of a productmay become possible. For example, an in vitro prosthetic kidney may beused to manufacture renin.

An embodiment of the invention is directed to the use of a prosthetickidney for the analysis of the effects of a substance on the kidney. Asubstance may be a drug or pharmaceutical, a chemical, a microbe, abiological product, or an element. A drug or pharmaceutical is anychemical compound that may be used on or administered to a patient,including humans and animals, as an aid in the diagnosis, treatment, orprevention of disease or other abnormal condition. A drug may be usedfor the relief of pain of suffering, or to control or improve anyphysiologic or pathologic condition. Examples of drugs, includevaccines, recombinant agents, chemicals, recombinant nucleic acid,recombinant protein, and living, dead, or attenuated microbes. Usefuldrugs for testing include candidate drugs, chemicals, compounds andagents which are suspected to have properties of a drug. Chemicals whichmay be tested may include any chemical or substance a patient or akidney may be exposed to. Such chemicals include environmentalchemicals, personal hygiene products and cosmetics. Microbes include anyliving organism such as bacteria, fungus, viruses, amoeba, parasites,and yeast or the like which may be living, dead, in suspended animation,quiescent or attenuated at the time of testing. Biologicals productsinclude products and waste products from living organisms such asproteins, lipids, nucleic acids, sugars, toxins which are produced froma living organism.

Treatment of Kidney Disease

In one embodiment of the invention, the prosthetic kidney may bemaintained and operated externally and function ex vivo. A patientrequiring blood treatment may connect their blood supply to theprosthetic kidney for a period of time. The treated blood may beseparated from the prosthetic kidney following treatment and returned tothe patient.

Another embodiment of the invention is directed to methods for treatingkidney disease by the augmentation of kidney function by implanting theprosthetic kidney in the patient. Kidney disease is a general term thatincludes diseases ranging from less life threatening diseases such askidney stones to more life-threatening disorders such as polycystickidney disease and nephrosis, temporary and chronic and permanent kidneyfailure. Diseases of the kidney which may be treated by the method ofthe invention include any disease which may benefit from augmentation ofrenal function such as congenital anomalies of the kidney such as,cystic renal dysplasia, polycystic kidney disease, cystic diseases ofrenal medulla, acquired (dialysis-associated) cystic disease and simplecysts; glomerular diseases such as, acute glomerulonephritis, crescenticglomerulonephritis, nephrotic syndrome, membranous glomerulonephritis,minimal change disease, lipoid nephrosis, focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, IgAnephropathy, focal proliferative glomerulonephritis, chronicglomerulonephritis, systemic lupus erythematosus, Henoch-Schonleinpurpura, bacterial endocarditis, diabetic glomerulosclerosis,amyloidosis and hereditary nephritis, tubule diseases such as acutetubular necrosis, acute renal failure, and other renal diseases such as,microangiopathic hemolytic anemia, atheroembolic renal disease, sicklecell disease nephropathy, diffuse cortical necrosis, renal infarcts,adenomas, carcinomas, nephroblastoma, immunologically mediated renaldisease, drug induced nephritis, urate nephropathy, hypercalcemia andnephrocalcinosis.

Other diseases which may be treated may comprise any conditions where apatient's kidney may be damaged. For example, the method of theinvention may be used to treat patients with healthy kidneys undergoingchemotherapy with a drug toxic to nephrons. Other conditions which mayrequire treatment include, for example, trauma, toxin ingestion,autoimmune disease, old age, and the like.

The method of the invention is useful for the treatment of any kidneydisease where it is desired to augment the function of a patient'snormal kidneys. The prosthetic kidney may be implanted for a limitedduration or permanently depending on whether the need for renalaugmentation is temporary or permanent.

The prosthetic kidney may be of many different shapes to fit the needsof the patient and the implantation site. For example, a prosthetickidney of an elongated or flat or compact shape may be optimal forabdominal or subcutaneous implantation where the patient's existingkidneys are not removed. Alternatively, a prosthetic kidney with theanatomic shape of a natural kidney may be most suited for patients whorequire kidney replacement. The size of the prosthetic kidney may alsobe varied for optimal performance. A larger patient may require a largerprosthetic kidney while a smaller patient, such as a child, may be moresuited to a smaller prosthetic kidney.

Other embodiments and advantages of the invention are set forth, inpart, in the description which follows and, in part, will be obviousfrom this description and may be learned from practice of the invention.

EXAMPLES Example 1 Isolation of Kidney Cells

Small kidneys and kidney sections of large kidneys, such as from oneweek old C57 black mice, were decapsulated, dissected, minced andsuspended in Dulbecco's Modified Eagles's Medium (DMEM; Sigma, St.Louis, Mo.) containing 15 mM Hepes, pH 7.4 and 0.5 μg/ml insulin, 1.0mg/ml collagenase and 0.5 mg/ml dispase, a neutral protease fromBacillus polymyxal (Boehringer Mannheim, Indianapolis, Ind.).

Large kidneys, such as swine kidneys, were arterially perfused at 37°for 10 minutes with calcium free Eagles minimum essential medium withinthree hours of extraction. The kidneys were then perfused with 0.5 mg/mlcollagenase (Type IV, Sigma, St. Louis, Mo.) in the same buffersupplemented with 1.5 mM MgCl₂ and 1.5 mM CaCl₂. The kidneys were thendecapsulated, dissected, minced and suspended in Dulbecco's ModifiedEagles's Medium (DMEM; Sigma, St. Louis, Mo.) containing 15 mM Hepes, pH7.4 and 0.5 μg/ml insulin, 1.0 mg/ml collagenase and 0.5 mg/ml dispase,a neutral protease from Bacillus polymyxal (Boehringer Mannheim,Indianapolis, Ind.).

The kidney cell suspension, from either large or small kidneys, wasgently agitated in a water bath for 30 minutes at 37° C. The cells andfragments were recovered by centrifugation at 50 g for five minutes. Thepellets were resuspended in DMEM containing 10% fetal bovine serum(Biowhittaker, Walkersville, Md.) to stop proteolysis, and the turbidsolution was passed through sterile 80 mesh nylon screens to eliminatelarge fragments. The cells were recovered by centrifugation and washedtwice with calcium free Dulbecco's Modified Eagles's Medium.

Example 2 In vitro Culturing of Kidney Cells Isolation of Rat TailCollagen

Tendon was stripped from rat tails and stored in 0.12 M acetic acid indeionized water in 50 ml tubes. After 16 hours at 4° C. overnight.

Dialysis bags were pretreated to ensure a uniform pore size and removalof heavy metals. Briefly, the dialysis bag is submerged in a solution of2% sodium bicarbonate and 0.05% EDTA and boiled for ten minutes.Multiple rinses of distilled water was used to remove the sodiumbicarbonate and 0.05% EDTA.

The 0.12 M acetic acid solution comprising rat tendons was placed intreated dialysis bags and dialyzed for two or three days to removeacetic acid. The dialysis solution was changed every 3 to 4 hours.

Treatment of Porous Membrane Structure With Collagen

A porous membrane structure (FIG. 4) was treated by contact with asolution containing about 30 μg/ml collagen (Vitrogen or rat tailcollagen), about 10 μg/ml human fibronectin (Sigma, St. Louis, Mo.) andabout 10 μg/ml bovine serum albumin (Sigma, St. Louis, Mo.) in a totalvolume of about 2 ml of supplemented medium by incubation at 37° C. for3 hours. Then the collagen coated porous membrane structure was placedinto an incubator with 1 ml concentrated ammonium hydroxide (about 28%to about 30% NH₄OH, Sigma, St. Louis, Mo.) for 30 minutes to raise thepH and to promote the gelling of the collagen. After ammonium hydroxidetreatment of the porous membrane structure, the structure was washedextensively with isotonic medium to neutralize the pH of the porousmembrane structure before use.

Coating Tissue Culture Plates

The culture flasks, 75 cm², were coated with a solution containing about30 μg/ml collagen (Vitrogen or rat tail collagen), about 10 μg/ml humanfibronectin (Sigma, St. Louis, Mo.) and about 10 μg/ml bovine serumalbumin (Sigma, St. Louis, Mo.) in a total volume of about 2 ml ofsupplemented medium by incubation at 37° C. for 3 hours.

Cell Culture

Digested single suspended renal cells were plated on a modified collagenmatrix at a concentration of about 1×10⁶ cells/ml and grown in DMEMsupplemented with about 10% fetal bovine serum, about 5 μg/ml bovineinsulin, about 10 μg/ml transferrin, about 10 μg/ml sodium selenite,about 0.5 μM hydrocortisone, about 10 ng/ml prostaglandin E₂, about 100units/ml penicillin G, about 100 μg/ml streptomycin (Sigma, St. Louis,Mo.) in a 5% CO₂ incubator at about 37 °.

Confluent monolayers were subcultured by treatment with about 0.05%trypsin, about 0.53 mM EDTA (Gibco BRL, Grand Island, N.Y.) in calciumion free phosphate buffer saline (PBS) (about 1.51 mM KH₂PO₄, about155.17 mM NaCl, about 2.8 mM Na₂HPO·7H₂O).

Cells may be cultured any time from the first passage by suspension inabout 10% DMSO in culture medium for freezing and storage in liquidmedium.

Example 3 Preparation of the Prosthetic Kidney

Kidney cells were cultured and expanded in vitro for 10 days. In vitroculture medium, DMEM supplemented with 10% fetal bovine serum, 5 μg/mlbovine insulin, 10 μg/ml transferrin, 10 μg/ml sodium selenite, 0.5 μMhydrocortisone, 10 ng/ml prostaglandin E₂, 100 units/ml penicillin G,100 μg/ml streptomycin (Sigma, St. Louis, Mo.), was changed every otherday. The cells were harvested by trypsin digestion using 0.05% trypsin,about 0.53 mM EDTA (Gibco BRL, Grand Island, N.Y.) in calcium ion freephosphate buffer saline (PBS) (about 1.51 mM KH₂PO₄, about 155.17 mMNaCl, about 2.8 mM Na₂HPO·7H₂O). After digestion for 10 minutes at 37°C. the cells were resuspended in DMEM media at approximately 5×10⁶cells/ml.

The cell suspension was gently layered onto a porous membrane structurecomprising a preformed tubular device constructed from polycarbonatemembrane with 4 micron pore size connected at one end with silasticcatheter leading into a reservoir. The porous membrane structure wascoated with rat collagen (Example 2). The porous membrane structure,layered with approximately 10⁷ cells per square centimeter of porousmembrane surface was incubated at 37° C. under 5% CO₂ for about 30minutes to about 40 minutes. At the end of the incubation period,additional prewarmed media, is gently added until the porous membranestructure is submerged. The porous membrane structure was incubated at37° C. under 5% CO₂ for about 7 days to about 10 days. Media was changedand the cells were fed at frequent intervals such as for example, aboutevery day, about every two days or about every three days.

At about seven to about 10 days after seeding, artificial renal unitprecursors developed on the surface of the porous membrane structure.After 30 days of in vitro culture, a fluid was observed and collected atthe reservoir of the porous membrane structure. While the in vitroculture media is red because of phenol red, the fluid in the reservoiris transparent and colorless. The collection of a fluid distinct fromthe media indicates that the artificial renal unit precursors exhibitfiltration or secretion functions.

Implantation of Artificial Renal Unit Precursors

After about 7 to about 10 days after seeding, some of the porousmembrane structures comprising artificial renal unit precursors on theirsurface were implanted in the subcutaneous space of athymic mice.Athymic mice may be purchased from commercially from suppliers such asJackson Laboratories of Bar Harbor, Me. Animals were sacrificed at abouttwo, about four, and about eight weeks post-implantation and theartificial renal unit precursors were retrieved and analyzed.

Retrieved specimens were examined grossly and histologically withhematoxylin and eosin. Inununohistochemical stains for osteopontin,fibronectin and alkaline phosphatase were performed to determine thecell types and their architecture in vivo (FIGS. 5, 6, 7). Humanfibronectin monoclonal antibody (Sigmna, St. Louis, Mo.) was usedagainst fibronectin matrix. Rhodarnine-conjugated goat anti-mouse(Boehringer Mannheim, Indianapolis, Ind.) was used as a secondaryantibody. Immunocytochemical staining for osteopontin (FIG. 5) wasperformed with a polyclonal antibody produced in our laboratory.Antibodies were produced in New Zealand white rabbits using standardprocedures (Harlow and Lane, Antibodies a laboratory manual, 1988, ColdSpring Harbor Press, Cold Spring Harbor) and used at a 1:5000 dilutionratio. Goat anti-rabbit antibody conjugated with FITC (BoehringerMannheim, Indianapolis, Ind.) was used as a secondary antibody.Immunohistochemical stain for alkaline phosphatase using nitrobluetetrazolium and 5-Bromo-4-choloro-3-indolyl phosphate (Sigma, St. Louis,Mo.) was performed. Filtrate collected from the prosthetic kidney wasstraw yellow in color. Analysis of the filtrate for uric acid level wasperformed using a uric acid detection kit (Sigma Diagnostics, St. Louis,Mo.).

All animals survived until the sacrifice. Retrieved specimens maintainedtheir original-architecture. The artificial renal units precursors werecovered by host tissue grossly. The fluid in the prosthetic kidney wascollected in the catheters connected to the membrane. Histologicalexamination of the implanted prosthetic kidneys revealed extensivevascularization, formation of glomeruli (FIG. 8) and highly organizedtubule-like structures. Immunocytochemical staining withanti-osteopontin antibody which is secreted primarily by proximal anddistal tubule cells stained the tubular sections positively.Immunohistochemical staining for alkaline phosphatase stained proximaltubule like structures positively. Furthermore, uniform staining forfibronectin in the extracellular matrix of newly formed tubules wasobserved (FIG. 6). The yellow fluid collected from the newly formedrenal unit contained 66 mg/dl uric acid, as compared to 2 mg/dl inplasma, suggesting that these tubules are capable of unidirectionalsecretion and concentration of uric acid. The evidence of glomerutiformation, the histological staining and the secretion of uric acidindicates that by 7 days post-implantation, the artificial renal unitprecursor has developed into an ARU.

Phenotypical Comparison Between Renal Tubule Analogs and Natural Tubules

Proximal tubule cells of the kidney are cuboidal in shape. They containa centrally placed round to oval nucleus with one or two prominentnucleoli. The apical surface of the cell is formed into long, slenderfinger-like microvilli which in animals such as the rat reach heights of1.3 mm and increase apical surface area approximately 22-fold. Incontrast the distal convoluted tubule usually contains short stubbymicrovilli on its apical surface and the collecting tubules lack a brushborder.

The cells of the renal tubule analog resemble natural kidney tubules.Artificial kidney tubule cells have an extensive brush border of longslender microvilli which extend into the surface invaginations. Themicrovillar density of cultured cells is comparable to that found inproximal tubule cells in vivo. The lateral and basal borders of theproximal cells are highly irregular and show extensive interdigitationswith adjacent cells similar to that observed in cultured cells.

One characteristic of proximal tubule cells is the presence of largenumbers of lysosomes, phagosomes and peroxisomes also seen in largenumbers in cultured cells. The maintenance of alkaline phosphatase andgamma glutamyltransferase activities, marker enzymes for the brushborder of proximal tubule cells, over long periods of culture providesfurther evidence for the identity and integrity of the artificial renaltubules.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All U.S. Patents and other referencesnoted herein for whatever reason are specifically incorporated byreference. The specification and examples should be considered exemplaryonly with the true scope and spirit of the invention indicated by thefollowing claims.

We claim:
 1. A method for making a nephron analog comprising the stepsof: (a) providing a porous membrane structure having an external surfacedefining an enclosed internal space having at least one effluentchannel; (b) contacting said external surface with a suspension ofkidney tissue cells; (c) culturing said kidney cells on said externalsurface in vitro to form a plurality of renal tubule analogs, eachhaving a region for glomeruli structure formation, said renal tubuleanalogs comprising a three-dimensional aggregate of kidney tubule cellshaving a brush border configured to contact host tissue to induceformation of the glomeruli structure and a lumen in fluid communicationwith the enclosed space of said membrane structure; and (d) implantingsaid membrane structure having attached to said external surface thereofa plurality of renal tubule analogs into a host, whereby said renaltubule analog contacts host tissue and induces said host to produceglomeruli structures in at least one region of said renal tubule analog,thereby making a nephron analog.
 2. The method of claim 1 wherein thebrush border comprises a plurality of microvilli on the free surface ofsaid kidney cell.
 3. The method of claim 1 wherein the cell aggregatesexpress osteopontin and the kidney tubule cells in at least one regionof said aggregate express alkaline phosphatase.
 4. The method of claim 1wherein the cell aggregate is positive on a Periodic Acid Schiffstaining assay.
 5. The method of claim 1 wherein the renal tubuleanalogs express fibronectin.
 6. The method of claim 1 wherein the kidneytubule cells are positive in a Periodic Acid Schiff assay.
 7. The methodof claim 1 wherein the kidney cells are fetal kidney cells or juvenilekidney cells.
 8. The method of claim 1 wherein the kidney cells are froma kidney cortex.
 9. The method of claim 1 wherein the kidney cells arehuman.
 10. The method of claim 1 wherein the enclosed porous membranestructure prevents passage of cells and permits passage of fluid andgas.
 11. The method of claim 1 wherein the enclosed porous membranestructure comprises pores of between about 0.04 micron to about 10microns in diameter.
 12. The method of claim 1 wherein the enclosedporous membrane structure comprises pores of between about 0.4 micron toabout 4 microns in diameter.
 13. A method for making a nephron analogcomprising the steps of: a) isolating kidney tissue; b) disassociatingsaid kidney tissue by enzymatic treatment to form a cell suspension; c)culturing said kidney cell suspension in vitro; d) treating an enclosedporous membrane structure with extracellular matrix protein; e)culturing said kidney cells on the treated exterior surface of theenclosed porous membrane to form renal tubule analogs, each having aregion for glomeruli structure formation, said renal tubule analogscomprising three-dimensional aggregates of kidney tubule cells having abrush border configured to contact host tissue to induce formation ofthe glomeruli structure and containing lumens within the interior ofsaid aggregates; and f) implanting said membrane structure havingattached to said external surface thereof a plurality of renal tubuleanalogs into a host, whereby said renal tubule analog contacts hosttissue and induces said host to produce glomeruli structures in at leastone region of said renal tubule analog, thereby making a nephron analog.14. The method of claim 13 wherein the extracellular matrix proteincomprises collagen.
 15. The method of claim 13 wherein the collagen israt tail collagen.
 16. The method of claim 13 wherein the brush bordercompromises a plurality of micrbvilli on the free surface of said kidneycell.
 17. The method of claim 13 wherein the cell aggregates expressosteopontin and the kidney tubule cells in at least one region of saidaggregate express alkaline phosphatase.
 18. The method of claim 13wherein the cell aggregate is positive on a Periodic Acid Schiffstaining assay.